Data Sheet: Sequencing
TruSight Cancer Sequencing Panel
®
Expert-defined content targeting genes associated with a predisposition for various cancers
delivered on proven next-generation sequencing technology.
Highlights
• Expert-Defined Content
Chosen by The Institute of Cancer Research, London to
target genes associated with a predisposition towards cancer
• Low Input DNA Requirement
Excellent data quality with as little as 50 ng DNA to preserve
precious samples
• Fast, Simple Workflow
Library preparation and enrichment completed in 1.5 days
Introduction
As we learn more about the role genetic variants play in cancer
predisposition, researchers will need to be able to evaluate the
genes in which these variants lie comprehensively. This process is
now simpler and faster with the TruSight Cancer Sequencing Panel.
Developed in collaboration with experts in cancer genomics, the
predesigned, ready-to-use oligos specifically target genes associated
with cancer. The sequencing panel is compatible with TruSight Rapid
Capture kits, providing a single, integrated library preparation and
enrichment workflow that can be completed in just 1.5 days (Figure 1).
Delivering excellent data quality from low sample input (50 ng),
TruSight Cancer and TruSight Rapid Capture enable researchers to
analyze precious samples, while retaining sufficient material for future
analyses.
Content Design Strategy
The TruSight Cancer Sequencing Panel was developed in collaboration
with Professor Nazneen Rahman and team at The Institute of Cancer
Research (ICR), London. It targets 94 genes suspected to play a
role in predisposing to cancer, including genes associated with both
common (eg, breast, colorectal) and rare cancers. In addition, the
panel includes 284 SNPs suspected to be associated with cancer
through genome-wide association studies (GWAS). Content selection
was based on expert curation of the scientific literature and other high-
quality resources.
Superior Coverage
TruSight Cancer features a highly optimized probe set that delivers
comprehensive coverage of genes suspected to indicate a
predisposition for cancer, starting from only 50 ng of DNA input.
The kit includes ~4,000 80-mer probes, each constructed against
the human 170–220 NCBI37/hg19 reference genome. The probe set
was designed to enrich for > 1,700 exons, spanning 94 genes of
interest (Table 1).
For Research Use Only. Not for use in diagnostic procedures.
Prepare indexed, pooled library from 50 ng of DNA
Capture targeted regions using
TruSight Rapid Capture and TruSight Cancer
Add enriched sample pool to MiniSeq® flow cell
and place on MiniSeq system for sequencing*
Analyze data: On-instrument Local Run Manager
software or the BWA Enrichment BaseSpace® App
automatically performs alignment and variant calling
Interpret data using reporting software
such as the VariantStudio
Figure 1: Integrated Workflow—TruSight Cancer is compatible with the
TruSight Rapid Capture method, which integrates library preparation and
enrichment steps. This offers a fast, streamlined, and optimized workflow,
delivering fully enriched libraries for up to 96 samples in just 1.5 days.
*TruSight Cancer is also compatible with the MiSeq®, NextSeq®, and HiSeq®
Series Systems.
The TruSight Cancer Sequencing Panel targets a total of 255 kb of the
human genome. The 80-mer probes target libraries of approximately
500 bp (insert size of 300 bp), enriching 350–650 bases centered
symmetrically around the midpoint of the probe (Figure 2)1. This means
that the kit provides coverage of exonic and noncoding DNA in exon-
flanking regions, on average 50 bp.
Integrated Library Preparation and
Enrichment Workflow
TruSight Cancer and TruSight Rapid Capture harness the speed of
Nextera® library preparation technology. It eliminates the need for
mechanical DNA fragmentation and introduces a unique multiplex
pre-enrichment sample pooling step. The TruSight Rapid Capture
method reduces hands-on time for a high-throughput workflow that
saves at least one full day over all other currently available enrichment
workflows (Figure 1). Furthermore, master-mixed reagents are coupled
with a plate-based protocol for simultaneous processing of up to 24
enrichment reactions (288 total samples).
Flexible kit configurations enable labs to meet their sample throughput
needs. For those requiring higher throughput, kit reagent volumes are
optimized for liquid handlers to make an automation-friendly workflow.
TruSight Rapid Capture kits supporting lower throughput options are
also available, allowing labs to run samples immediately instead of
waiting to batch.
Data Sheet: Sequencing

Table 1: Coverage Details Library size (~330 bp)

Cumulative target region size 255 kb Target (150 bp)
Number of target genes 94 Adapter (~65 bp) Insert ~20 bp Adapter (~65 bp)

Number of target exons > 1700

80-mer probe
Probe size 80-mer
Number of probes ~ 4000 > 60% of total reads map to target region
Recommended mean coverage 100×
Figure 2: Probe Footprint—With an approximately 500 bp DNA library (insert
Target minimum coverage 20× size of 300 bp), the probe will enrich 250–650 bp centered on its midpoint.
Percent exons covered based on coverage metrics ≥ 95%

Transposomes Genomic DNA Biotin probes

~ 300 bp

Tagmentation

C. Hybridize biotinylated probes to targeted regions

~ 300 bp

P5
Read 2 Sequencing Primer
Index 1
Index 2
Read 1 Sequencing Primer
P7 Streptavidin beads
PCR Amplification

Enrichment-Ready Fragment

A. Sample Preparation

D. Enrichment using streptavidin beads

Pooled Sample Library

Sequencing-Ready Fragment

B. Denature double-stranded DNA library (for simplicity, adapters E. Elution from beads
and indexes not shown)

Figure 3: Integrated TruSight Rapid Capture Workflow—The TruSight Rapid Capture workflow provides a fast, simple method for isolating the genes targeted using
TruSight Cancer. The streamlined, automation-friendly workflow combines library preparation and enrichment steps, and can be easily completed in 1.5 days with
minimum hands-on time.

For Research Use Only. Not for use in diagnostic procedures.

 Data Sheet: Sequencing

90
Percent of Regions Covered

80

70

60

50 0.2× Mean Coverage

40 0.5× Mean Coverage

1.0× Mean Coverage

30

20

10

0
1 2 3 4 5 6 7 8 9 10 11 12

Samples

Figure 4: High Coverage Uniformity— Coverage uniformity is given for 12 samples regarding the percentage of targeted regions at varying mean normalized read
depths. The 12 samples were prepared and then simultaneously enriched using the TruSight Rapid Capture along with the TruSight Cancer Sequencing Panel. Pooled
samples were sequenced across a MiSeq® standard flow cell, generating mean read depths of 130–230× (varying for each sample). Over 95% of bases (~ 250 kb)
were covered at 0.2× mean coverage.

The process starts with rapid Nextera-based library prep to convert
input genomic DNA into adapter-tagged libraries (Figure 3A). This rapid
prep requires only 50 ng of input DNA and takes less than 3 hours for
a plate of 96 samples. Nextera tagmentation of DNA simultaneously
fragments and tags DNA without the need for mechanical shearing.
Integrated sample barcodes then allow the pooling of up to 96
samples for a single Rapid Capture pulldown. Next, libraries are
denatured into single-stranded DNA (Figure 3B) and biotin-labeled
probes specific to the targeted region are used for the Rapid Capture
hybridization (Figure 3C). The pool is enriched for the desired regions
by adding streptavidin beads that bind to the biotinylated probes
(Figure 3D). Biotinylated DNA fragments bound to the streptavidin
beads are magnetically pulled down from the solution (Figure 3E).
The enriched DNA fragments are then eluted from the beads and
hybridized for a second Rapid Capture. This entire process is
completed in only 1.5 days, enabling a single researcher to process up
to 288 samples at one time—all without automation.
Data Analysis
Sequence data generated from TruSight Cancer enriched libraries are
analyzed by the on-instrument Local Run Manager software or the
BWA Enrichment BaseSpace App. After demultiplexing and FASTQ file
generation, the software uses the Burrows-Wheeler Aligner (BWA) to align
the reads against the hg19 homo sapiens reference genome to create
BAM files. The Genome Analysis Toolkit (GATK) is then used to perform
variant analysis for the target regions specified in the manifest file. The
output of GATK are VCF files, which are text files that contain SNPs,
indels, and other structural variants. Summary tables are generated to
report on enrichment, variant calling, coverage, insert fragment length,
and duplicates.
High Data Quality
With TruSight Cancer and TruSight Rapid Capture, researchers can
be confident in the quality of sequencing data generated from pooled
multisample libraries. Each sample is sequenced with high coverage
uniformity across the target region, with 95% of exons covered at a
minimum coverage of 20× (Figure 4). This uniformity applies to smaller
exons (< 150 bp) and long coding exons.
Summary
The TruSight Cancer Sequencing Panel enables researchers to access
an expert-defined content set for analyzing variation within genes
previously linked with a predisposition towards cancer. The optimized
probe set provides comprehensive coverage of the targeted regions
with high coverage uniformity for identifying many variants. Combining
this content with the TruSight Rapid Capture method enables a fast,
easy workflow, requiring low sample DNA input, generating a highly
efficient resequencing solution to accelerate detection of genes
associated with cancer.
Learn More
To learn more about TruSight Cancer Sequencing Panel, TruSight
Rapid Capture kits, and Illumina next-generation sequencing
technology, visit www.illumina.com/trusightcancer.
References
1. Optimizing Coverage for Targeted Resequencing Technical Note (www.
illumina.com/documents/products/technotes/technote_optimizing_
coverage_for_targeted_resequencing.pdf)

For Research Use Only. Not for use in diagnostic procedures.

Data Sheet: Sequencing

Ordering Information

Product Catalog No. TG Catalog No.*

TruSight Cancer Sequencing Panel (4 enrichments) FC-121-0202 TG-141-1002
Rapid Capture Kits
TruSight Rapid Capture (1 index, 8 samples) FC-140-1101 TG-140-1101
TruSight Rapid Capture (2 indexes, 8 samples) FC-140-1102 TG-140-1102
TruSight Rapid Capture (4 indexes, 16 samples) FC-140-1103 TG-140-1103
TruSight Rapid Capture (24 indexes, 48 samples) FC-140-1104 TG-140-1104
TruSight Rapid Capture (24 indexes, 96 samples) FC-140-1105 TG-140-1105
TruSight Rapid Capture (96 indexes, 288 samples) FC-140-1106 TG-140-1106
*TG-labeled consumables include features intended to help customers reduce the frequency of revalidation. They are available only under supply agreement and require customers to provide a
binding forecast. TruSight sequencing panels are available for evaluation purposes before executing a supply agreement. Please contact your account manager for more information.

Note regarding biomarker patents and other patents unique to specific uses of products.

Some genomic variants, including some nucleic acid sequences, and their use in specific applications may be protected by patents. Customers
are advised to determine whether they are required to obtain licenses from the party that owns or controls such patents in order to use the product
in customer's specific application.

Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com

For Research Use Only. Not for use in diagnostic procedures.
© 2016 Illumina, Inc. All rights reserved. Illumina, TruSight, MiniSeq, MiSeq, NextSeq, HiSeq, BaseSpace, Nextera, and
the pumpkin orange color are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries.
Pub. No. 670-2012-007 Current as of 08 January 2016